A cobalt porphyrin containing protein reducible by hydrogenase isolated from Desulfovibrio desulfuricans (Norway)

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Vol. 103, No. 2,198l November

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30, 1981

RESEARCH COMMUNICATIONS Pages 521-530

A. COBALTPORPHYRIN CONTAININGPROTEINREDUCIBLE BY HYDRo(;ENAsE

ISCLATBDFROMDESULFOVIBRIO DESULFURICANS (Norway) E.C. Hatchikian Laboratoire de Chimie BactBrienne, C.N.R.S., 13274 Marseille Cedex 2, France Received

October

5.1981

A cobalt-porphyrin containing protein has been isolated fran the sulfate-reducer Desulfovibrio desulfuricans (Norway). This violet-colored protein has a molecular weight of approx. 13,GOO ,daltcns and contains 1 cobalt atcm/molecule. The apo-protein was estimated to contain 104 amino-acid residues giving a molecular weight of 11,000 daltons. The W-visible absorption spectrwn of the protein exhibiting maxima at 588,418 and 280 nm with a shculder at 550 nm is characteristic of metalloporph~in proteins. The molar extinction coefficients of the cobalt-protein at 588, 418 and 280 nm are 31,330 , 64,670 and 17,200 respectively and its absorbance ratio is 0.54 . The protein is reduced by dithionite giving a bluereduced form. Important spectral modifications of the chromophore occurred during the reduction including a shift of the Soret peak from 418 to 381 nm and a shift of the a band in the opposite direction fran 588 to 593.5 nm. The Co-protein was slowly reduced by the hydrogenase fran D.desulfuricans under hydrogen in the presence of cytochrcane c . The reporma suggest that the redox states of the cobalt center of t&is new electron carrier correspond to the Co(III) and Co(I1) states. INl’RODUCTION Sulfate-reducing

bacteria

are present day representatives

ancient process : the dissimilatory exhibit

an intricate

redox proteins

electron-transfer

(1,Z) . Several c-type

well as proteins with different lated fran Desulfovibrio these electron

reduction of sulfates.

transfer

redox properties Desulfovibrio

system constituted cytochromes, different

types of iron-sulfur

species (2,3).

of a very

Hover, by highly

they complex

flavoproteins

clusters have been iso-

Physico-chemical characterization

proteins have allowed a better

as

of

understanding of their

(2). desulfuricans

Noway

strain

is replaced by another pigment (desulforubidin)

lacks desulfoviridin catalyzing

(4) which

the dissimilatory

0006-291X/81/220521-10$01.00/0 521

Copyright 0 1981 by Academic Press, Inc. AN rights of reproduction in any form reserved.

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Vol. 103, No. 2,198l

reduction of bisulfite

AND

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(5). In addition to sulfate,

able to use elemental sulfur

as electron

doxin (7) have been characterized work deals with the isolation

ferredoxins

from D.desulfuricans

and the characterization

to the cobalt-containing

protein

trans-

(7,9) and rubre-

(Norway). The present of a protein

from this microorganism. This violet-colored

appears to be similar D.gigas (10)

this microorganism is

acceptor (6). Several electron

fer proteins including c-type cytochromes (7,8),

a cobalt-porphyrin

RESEARCH COMMUNICATIONS

recently

containing

protein which isolated from

is reducible by the hydrogenase-cytochrome c3 system under hydro-

gen atmosphere .

M4TERIALSANDMBIWDS Analytical methods. D.desulfuricans Norway 4 (NCIB 8310) was grown at 35’C on lactate-sulfate mediumas reported previously (7). Analytical gel electrophoresis was performed in 7 per cent polyacrylamide gel with Tris-HCl glycine buffer at pH 8.8 . SDS-polyacrylamide gel electrophoresis was carried out using the method of Weber and Osborn (11). The molecular weight of the protein was determined by gel filtration on a Sephadex G-75 column following the procedure of Whitaker (12). It was also estimated by the method of Weber and Osborn (11) on SDS-gel electrophoresis using the following standards : ovalbumin (43,000)) chymotrypsinogen (25,000) soybean trypsin inhibitor (20,100) and horse heart cytochrome c (12,500). Protein was determined according to the procedure of Lowry (13) or estimated from amino acid analysis. Amino acid analysis were performed on a LKB 3201 amino acid analyzer. Protein samples were hydrolyzed for 18 , 24 and 48 h at 110°C in 6 N HCl according to the method of Moore and Stein (14). The average was calculated from several analysis. Cysteine and methionine were analyzed after performic acid oxydation as cysteic acid and methionine sulfone, respectively, according to Hirs (15) . Cobalt and iron in the protein were estimated by attic absorption spectrcmetry using an Unicam model SP 1900 spectrameter. Absorption spectra were measured on a Cary 14 spectrophotometer. Molar extinction coefficients of the protein were obtained by measuring the values of the optical densities at its absorption maximausing a solution of known protein concentration calculated from amino acid analysis. The reduction of the cobalt-containing protein has been carried out under anaerobic conditions using suitable cuvettes with rubber stoppers. The protein and buffer have been bubbled with argon. The reaction started after injection of dithionite. The reduction of the Co-protein by the hydrogenase-cytochro c3 system has been performed under similar anaerobic conditions. The reaction started after flushing the cuvette with hydrogen as described previously (16). A pure hydrogenase preparation isolated from D.desulfuricans (Norway) and exhibiting a specific activity of 1830 umoles H evolved/min/mg protein (Hatchikian E.C., unpublished results) has been utiliz izd in the electron hransfer reactions. The cytochrome c used in the redox studies was purified from D.desulfuricans (Norway) as prev&usly reported (7). Isolation of the Cobalt-protein from D.desulfuricans. The bacterial paste (1,500 g wet weight) was suspended in 600 ml of 10 m Tris-HCl buffer con-

522

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Vol. 103, No. 2,198l

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taining a few deoxyribonuclease crystals and the cell suspension was passed once through a French pressure cell. The crude extract was prepared by centrifuging the resulting extract at 38,000 x g for 30 min. The acidic protein extract which contains the Co-protein and the hydrogenase activity rubredoxin and desulforubidin was subsequently in addition to ferredoxins, obtained as previously reported (7). The acidic protein extract (990 ml) was dialysed overnight and adsorbed onto two DEE-cellulose (WhatmanDE&E 52) columns (4 x 24 cm) previously equilibrated with 10 I&! Tris-HCl and eluted with a discontinuous gradient of the samebuffer (50 + 600 n&l). A fraction containing the Co-protein with desulforubidin and hydrogenase was eluted with ZOO-260mMTris-HCl buffer. After dialysis, this fraction was adsorbed on a second DE/E-cellulose column (4.2 x 18 cm) and eluted with a discontinuous Tris-HCl gradient from 50 to 350 n&l using 150 ml of each TrisHCl molarity. The fraction containing the Co-protein as well as the hydrogenase was eluted with ZOO-280ti and collected in a volume of 340 ml. It was then concentrated to 120 ml in a 350 ml ultrafiltration cell with a PM10 filter (Amicon). The subsequent fraction was divided in two parts of 60 ml and each filtered through an Ultrogel AcA 44 column (5 x 100 cm). At this stage, the Co-protein which appears on the column as a violet-colored band was separated from hydrogenase and desulforubidin. The protein was then applied on a HA-Ultrogel (IBF) column (2.5 x 8 cm) equilibrated with 20 n&l Tris-HCl. The Co-protein was not adsorbed on the column and collected in a volume of 150 ml. The resulting fraction was concentrated on a small DWcellulose column and subsequently passed through an Ultrogel AcA 54 column (2.5 x 100 an). Finally the Co-protein was adsorbed on a hydroxylapatite (HTP, Bio-Pad) CO~UITUI (2 x 6 cm) and eluted with 10 mMphosphate buffer. At this stage, the protein which exhibited an intense violet colour, was judged to be pure both from its spectrum (Azso/Asss = 0.54) and its amino acid composition. The yield was approx. 10 mg. RESULTS Homogeneity of the protein trophoresis,

the purified

a violet-colored tion to the first.

- Whensubjected to polyacrylamide

Co-protein showsbefore staining

band. After

staining

The possibility

slightly

pattern,

indicating

the presence of

the gels, a small band appears in addi-

that this second band could be the apo-

protein was checked with SDS-gel electrophoresis. electrophoretic

gel elec-

The results showed the same

that the second band has a molecular weight

lower than that of the main band, When the Co-protein was treated

with 2 N HCl and heated at 80’ for IO min, its SDS-gel electrophoresis

showed

the presence of a single band corresponding to the apo-protein. Molecular weight - The molecular weight of the Co-protein was estimated to be 13,000 daltons by gel filtration

on Sephadex G-75. The SDS-gel electro-

phoresis indicated molecular weights of 13,500 and 12,000 for the native protein and the apo-protein

respectively.

The minimummolecular weight deter-

mined from the amino acid composition was 11,001

523

without the prosthetic

group.

Vol. 103, No. 2,198l

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Amino acid composition - The amino acid composition of the Co-protein is reported in Table I. The protein was estimated to contain 104 residues. The comparison of its amino acid composition with that of D. gigas Co-protein shows that the two proteins contain a number of acidic residues higher than the basic ones. They exhibit clearly

by their

both a high amount of methionine but differ

content in cysteine,

valine,

leucine and aromatic residues.

Metal analysis - The protein was analyzed for iron and cobalt by atomic absorption spectrometry.

Cobalt was the only metal that was detected. An

average value of 0.9 cobalt atoms per molecule was obtained from three samples of the protein. Absorption spectrum and extinction tion spectrum of D. desulfuricans

coefficients

- The W-visible

absorp-

Co-protein is shown in Figure 1. It exhibits

absorption maxima at 588, 418 and 280 nmwith shoulders at about 550, 396 and are 0.54 and 2.06 resand A418'AS88 coefficients of the Co-protein at 588, 418

296 nm. The absorbance ratios A280'AS88 pectively.

The molar extinction

and 280 nm are 31,330 , 64,670 and 17,200 respectively tion spectrum of D. desulfuricans

cobalt-protein

and Soret (418 nm) bands is typical the spectra of D. desulfuricans

(Table I).

The absorp-

with e (588 run), B (550 run)

of metalloporphyrin

proteins

(17) .Although

and D. gigas Co-proteins are similar,

diffe-.

rences are apparent on the wavelenghts of maxima absorption (Soret peak and protein peak) as well as on the values of the extinction I).

The reduction of D. desulfuricans

performed under anaerobic conditions. The protein

Co-protein by dithionite

approx. 3 hours. The reduced protein exhibits

shifted direction

(Table

has been

The results are reported in Figure 1.

reacted slowly with dithionite,

modifications

coefficients

the reduction being complete in a blue color.

Important spectral

of the chromophore occurred during the reduction

from 588 to 593.5 nm whereas the Soret peak shifted

: the u band

in the opposite

from 418 to 381 nm with an important absorption decrease. The

spectra recorded at different

stages of reduction exhibited

points occurring at 593.5 , 532 , 437 and 392 nm (Figure 1).

524

four isosbestic The spectra of

BIOCHEMICAL

Vol.103,No.2,1981

Table

I. Comparison

AND

of the physico-chemical

D.desulfuricans Amino acid

BIOPHYSICAL

characteristics

and D.gigas

(Norway)

RESEARCH COMMUNICATIONS

Co-proteins.

composition

LYS His

5

D.gigasa 7

2

2

Arg ASP TinSer Glu

3

5

10

9

7

8

D. desulfuricans

5

9

10

11

6

8

QY

11

16

Aki

73

13

PI-0

Cys (Half)

0

4.

Val

8

15

Met

8

6

Ile Leu

5

4

5

9

1

2

5

8

Vr Phe Total

residues

104

Molecular weight Cobalt atoms/molecule

11,ooP

16,69gb,

1

1

Spectral data lilolar extinction

coefficients

588

(M -1

136

cm-1 , at the indicated 22,000

550

12,130

8,830

418

64,670 42,280 17,200

280

14,900

278

a) From Moura et al. b) Minimum molecular

oxidized

and reduced

of cobalt

as chelated

to shift

to the blue

could

wavelenghts)

31,330

420

the

of

be observed

forms

metal upon

after

(10)

weight

of the protein

since reduction

anaerobic

calculated

the Soret

from amino acid composition.

are

indicative

Peak of cobalt

(18).

The reversibility

addition

of ferricyanide

525

of the presence porphyrin

is

known

of the reduction to the

reduced

BIOCHEMICAL

Vol. 103. No. 2.1981

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RESEARCH COMMUNICATIONS

0.1-

t 381

I

L

I

I

L 510

481

WAVE LENGTH

1

1

I CM

nm

F$ure 1 - Absorption spectra of oxidized and reduced forms of D.desulfurlcans Co-protein. A lcm light path stoppered cwette (total volume, lml) contaIned 15 @I of Co-protein in 20 n&l Tris-HC1 buffer ($I 7.6) under an atmosphere of argon. After addition of 200 nmoles of sodium dithionite the spectrum of the solution was recorded at regular intervals on a Gary 14 spectrophotometer. (-) spectrum of the oxidized protein. (-e-f spectrum of the protein 5 min after addition of dithionite. The following spectra (---) (...) were recorded respectively 1 and 3 hours after addition of dithionite,

protein.

The native

detected

spectral

Reduction Hydrogenase under

nase.

nite,

frun

modification

atmosphere.

are

a difference

has its appeared

maximum

is

failed

for

ferricyanide

of the

; the only

ct band to 586 nm.

to reduce

As reported the

compared

is observed

for

reduction

other

2. When the

to that

including

directly electron

526

the Co-protein carriers

spectrum

of the protein

a shoulder

(19,16) by hydroge-

of the Co-protein reduced

of the Soret

of 381 nm. Further notably

c3 system

of the Co-protein

on the wavelenght

at 386 nm instead

in the W region

shift

with

by the hydrogenase-cytochro

shown in Figure

by hydrogenase

also

is the

D. desulf’uricans

c3 was required

The results

reduced

reacted

of the Co-protein

hydrogen

cytochrome

Co-protein

spectral at about

by dithio-

peak which features 360 nm and

Vol.

103, No.

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nm

Figure 2 - Reduction of D.desulfuricans Co-protein by the hydrogenasecytochromec systemunder hydrogen. A lcm light path stoppered cuvette (total vosume1 m1) contained 13 nMof Co-protein, 0.12 UMof hydrogenaseand 0.04 PMof cytochromec in 20 n&iTris-HCl buffer (pH 7.6). (--) spectrumunder argon. (... f samesampleunder hydrogen (1 atm.) after 6 hours.

an increase of absorbance in the tions,

the cobalt-porphyrin

280

nm region.

containing protein

In our experimental condi-

showed low reactivity

towards

the hydrogenase-cytochrome c3 system since the complete reduction occurred after

approx.

dithionite

6

hours. The rate of the electron

transfer

reaction both with

and hydrogenase appeared to be very low suggesting that the cobalt

center is protected. DISCUSSION_

The cobalt-containing protein

protein

of low molecular weight which exhibits

with c1 , (? and y bands typical which could be dissociated not covalently 588

isolated from D. desulfuricans a visible

of a metalloporphyrin.

from the protein

bound to the protein.

The cobalt-porphyrin and heating is

of the a band in the

- 593 nm range of the spectnrm, its high extinction

527

absorption spectrum

by acidification

The position

is an acidic

coefficient

and the

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Vol. 103, No. 2,198l

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very small absorbance ratio A4,8 (~)/A588 the porphyrin

to which the cobalt

tetrahydroporphyrin,

(a)

RESEARCH COMMUNICATIONS

suggest very strongly

is bound is similar

atom

to a dihydro- or

like in heme d or siroheme or to a porphyrin a (17,

However we could not find in the literature

20-22).

that

any spectroscopic data

about Co-porphyrin complexes of these types. On the other hand, we can exclude the prosthetic tero-

group examined here to be a Co-porphyrin of the deu-

, meso- , proto-

(23) or copro- types (18, 24, 25). Indeed, in such

cases a and @ bands have comparable intensities nm range and the Y/a this violet-colored cently

absorbance ratio protein

and lie in the 520-570

lies between 10 and 20. Although

appears to be similar

isolated from _D. gigas (10)

compared to this homologousprotein.

to the Co-protein re-

it showssignificant

differences

The two proteins clearly

amino acid composition, spectral data and redox properties. thy that the values of the extinction

coefficient

differ

when by their

It is notewor-

of D.desulfuricans

Co-

protein at 588, 550 and 418 nm are approximately 30 per cent higher than those of D. gigas protein. proteins concerns their D.gigas, the protein

The most important difference

redox behaviour.

In contrast

isolated from D.desulfuricans

te and the reaction is reversed by ferricyanide.

between the two

to the Co-protein of is reduced by dithioni-

Furthermore, this Co-pro-

tein could be reduced by hydrogenase under hydrogen, however, this electron transfer

reaction requires catalytic

amounts of cytochrame c3 as reported

for the other non-heme containing electron 19). The Co-porphyrin containing protein to function

as an electron carrier,

carriers

of Desulfovibrio

from D.desulfuricans

(16,

appears thus

however, the redox state of the cobalt

center in the protein needs more investigation.

Although cobalt in its Co(I1)

state (d7) is expected to give rise to an EPR spectrum (26, 27) the redox state of the cobalt in the protein

is still

undetermined since no EPR spec-

trum could be detected at 77 K, 9.1 (312 from 90 PM samples of the oxidized or reduced protein ble oxidation

(Y. Henry, personnal conrnunication).

states of the cobalt atom ( Co(I)

528

Of the three possi-

, Co(I1) , Co(III)

), the

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Vol. 103, No. 2,198l

Co(I)

state

BIOPHYSICAL

(d8) is very unusual and unstable,

complex (28). So, despite ted for the Co(I1) duced protein progress

AND

to elucidate

the porphyrin

present

in the vitamin

B,2s

the lack of evidence from an EPR spectrum expec-

(d7) state,

correspond

like

RESEARCH COMMUNKATIONS

it is suggested that the oxidized

to the Co(II1)

and Co(II)

the redox state of the cobalt

states,

Studies

and reare in

center and to identify

in the protein.

ACKNOWLEDGEMENTS The author wishes to express his thanks to Dr. M. Bruschi of this Institute for the determination of the amino acid composition of the protein and to Mrs. N. Forget and G. Bovier-Lapierre for their skilful technical help. He is also grateful to Dr. Y. Henry, Institut de Biologie PhysicoChimique of Paris, for valuable discussion and critical reading of the manuscript.

1. Le gall,

lOth, 2. Le Gall, Topics 3. Xavier,

E.C. (1975) Proc. FBBS Meet. J., Bruschi, M. and Hatchikian, 40, 277-285. J., Der Vartanian, D.V. and H.D. Peck, Jr. (1979) Current in Bioenergetics, 9, 237-265. A.V., Moura, J.J.G. and Moura, I. (1981) Structure and Bonding

43, '187-213. 4. Miller, J.D.A. and Saleh, A.M. (1964) J. Gen. Microbial. 37, 419-423. 5. Lee, J.P., Yi, C., Le Gall, J. and H.D. Peck, Jr. (1973) J. Bacterial. 115, 453-455. 6. Biebl, H. and Pfenning, N. (1977) Arch. Microbial. 112, 115-117. E.C., Golovleva, L.A. and Le Gall, J. (1977) 7. Bruschi, M., Hatchikian, J. Bacterial. 129, 30-38. 8. Fauque, G., Bruschi, M. and Le Gall, J. (1979) Biochem. Biophys. Res. commun. 86, 1020-1029. 9. Guerlesquin, F., Bruschi, M., Bovier-Lapierre, G. and Fauque, G. (1980) Biochim. Biophys. Acta 626, 127-135. 10. Moura, J.J.G., Moura, I., Bruschi, M., Le Gall, J. and Xavier, A.V.

(198(J) Biochem. Biophys.

Res. Connrun. 92, 962-970.

11. Weber, K. and Osbom, M. (1969) J. Biol. Chem., 244, 4406-4412. 12. Whitaker, J.R. (1963) Anal. Chem., 35, 1950-1953. R.J. (1951) 13. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, J. Bio. Chem., 193, 265-275. 14. Moore, S. and Stein, W.H. (1963) In Methods in Enzymology (Colowick, S.P. and Kaplan, NO., eds.) vol. 6, pp. 819-831, Academic Press. Hirs, C.H.W. (1976) In Methods in Enzymology (Hirs, C.H.W., ed.) vol. 11 15.

pp. 167- 199, Academic Press, New York. M., Hatchikian, E.C. and Benlian, D. (1980) Biochem. Biophys. Res. Cormsun. 92, 1258-1265. 17. Falk, .J.E. (1964) In Porphyrins and Metalloporphyrins, Elsevier, New York. R.A., Hoffman, L., Suzuki, K. and Ferrari, A. 18. Tsutsui, M., Velapoldi, (1969) J. Am. Chem. Sot. 91, 3337-3341. 16. R'zaigui,

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19. Bell, 60,

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Peck, H.D., Jr. and Le Gall,

J. (1978) Biochimie

315-320.

20. Lemberg, R. and Barret, J. (1973) In Cytochromes, Academic Press. 21. Mx-phy, M.J., Siegel, L.M., Tove, S.R. and Kamin, H. (1974) Proc. Nat. Acad. Sci. USA 71, 612-616. 22. Smith, K.M. (1975) In Porphyrins and metalloporphyrins (Smith, K.M. ed.) 23. Yonetani, T., Yamamoto, H. and Woodrow, G.V., III (1974) J. Biol. Chem.

249, 682-690. 24. Sone, N. (1971) J. Biochem. 69, 753-760. 25. Fujii, K., Tanaka, A. and Fukui, S. (1974) Arch. Microbial. 97, 259-271. 26. Lin, W.C. (1979) In the Porphyrins IV B, pp. 355-377 (D. Dolphin, ed.) Academic Press. 27. Subramanian, J. (1975) In Porphyrins and Metalloporphyrins, pp. 553-589 (K.M. Smith, ed.) Elsevier. 28, Schrauzer, G.N. (1969) Ann. N.Y. Acad. Sci. 158, 526-539.

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